This invention relates to novel nucleotide sequences, polypeptides encoded thereby, vectors and host cells and host organisms comprising one or more of the novel sequences, and to a method of altering one or more characteristics of an organism. The invention also relates to starch having novel properties and to uses thereof.
Starch is the major form of carbon reserve in plants, constituting 50% or more of the dry weight of many storage organsxe2x80x94e.g. tubers, seeds of cereals. Starch is used in numerous food and industrial applications. In many cases, however, it is necessary to modify the native starches, via chemical or physical means, in order to produce distinct properties to suit particular applications. It would be highly desirable to be able to produce starches with the required properties directly in the plant, thereby removing the need for additional modification. To achieve this via genetic engineering requires knowledge of the metabolic pathway of starch biosynthesis. This includes characterisation of genes and encoded gene products which catalyse the synthesis of starch. Knowledge about the regulation of starch biosynthesis raises the possibility of xe2x80x9cre-programmingxe2x80x9d biosynthetic pathways to create starches with novel properties that could have new commercial applications.
The commercially useful properties of starch derive from the ability of the native granular form to swell and absorb water upon suitable treatment. Usually heat is required to cause granules to swell in a process known as gelatinization, which has been defined (W A Atwell et al, Cereal Foods World 33, 306-311, 1988) as xe2x80x9c . . . the collapse (disruption) of molecular order within the starch granule manifested in irreversible changes in properties such as granular swelling; native crystallite melting, loss of birefringence, and starch solubilization. The point of initial gelaltinization and the range over which it occurs is governed by starch concentration, method of observation, granule type, and heterogeneities within the granule population under observationxe2x80x9d. A number of techniques are available for the determination of gelatinization as induced by heating, a convenient and accurate method being differential scanning calorimetry, which detects the temperature range and enthalpy associated with the collapse of molecular orders within the granule. To obtain accurate and meaningful results, the peak and/or onset temperature of the endotherm observed by differential scanning calorimetry is usually determined.
The consequence of the collapse of molecular orders within starch granules is that the granules are capable of taking up water in a process known as pasting, which has been defined (W A Atwell et al, Cereal Foods World 33, 306-311, 1988) as xe2x80x9c . . . the phenomenon following gelatinization in the dissolution of starch. It involves granular swelling, exudation of molecular components from the granule, and eventually, total disruption of the granulesxe2x80x9d. The best method of evaluating pasting properties is considered to be the viscoamylograph (Atwell et al, 1988 cited above) in which the viscosity of a stirred starch suspension is monitored under a defined time/temperature regime. A typical viscoamylograph profile for potato starch shows an initial rise in viscosity, which is considered to be due to granule swelling. In addition to the overall shape of the viscosity response in a viscoamylograph, a convenient quantitative measure is the temperature of initial viscosity development (onset). FIG. 1 shows such a typical viscosity profile for potato starch, during and after cooking, and includes stages A-D which correspond to viscosity onset (A), maximum viscosity (B), complete dispersion (C) and reassociation of molecules (or retrogradation, D). In the figure, the dotted line represents viscosity (in stirring number units) of a 10% w/w starch suspension and the unbroken line shows the temperature in degrees centigrade. At a certain point, defined by the viscosity peak, granule swelling is so extensive that the resulting highly expanded structures are susceptible to mechanically-induced fragmentation under the stirring conditions used. With increased heating and holding at 95xc2x0 C., further reduction in viscosity is observed due to increased fragmentation of swollen granules. This general profile has previously always been found for native potato starch.
After heating starches in water to 95xc2x0 C. and holding at that temperature (for typically 15 minutes), subsequent cooling to 50xc2x0 C. results in an increase in viscosity due to the process of retrogradation or set-back. Retrogradation (or set-back) is defined (Atwell et al., 1988 cited above) as xe2x80x9c . . . a process which occurs when the molecules comprising gelatinised starch begin to reassociate in an ordered structure . . . xe2x80x9d. At 50xc2x0 C., it is primarily the amylose component which reassociates, as indicated by the increase in viscoamylograph viscosity for starch from normal maize (21.6% amylose) compared with starch from waxy maize (1.1% amylose) as shown in FIG. 2. FIG. 2 is a viscoamylograph of 10% w/w starch suspensions from waxy maize (solid line), conventional maize (dots and dashes), high amylose variety (HYLON(copyright) V starch, dotted line) and a very high amylose variety (HYLON(copyright) VII starch, crosses). The temperature profile is also shown by a solid line, as in FIG. 1. The extent of viscosity increase in the viscoamylograph on cooling and holding at 50xc2x0 C. depends on the amount of amylose which is able to reassociate due to its exudation from starch granules during the gelatinization and pasting processes. A characteristic of amylose-rich starches from maize plants is that very little amylose is exuded from granules by gelatinization and pasting up to 95xc2x0 C., probably due to the restricted swelling of the granules. This is illustrated in FIG. 2 which shows low viscosities for a high amylose (44.9%) starch (HYLON(copyright) V starch) from maize during gelatinization and pasting at 95xc2x0 C. and little increase in viscosity on cooling and holding at 50xc2x0 C. This effect is more extreme for a higher amylose content (58%, as in HYLON(copyright) VII starch), which shows even lower viscosities in the viscoamylograph test (FIG. 2). For commercially-available high amylose starches (currently available from maize plants, such as those described above), processing at greater than 100xc2x0 C. is usually necessary in order to generate the benefits of high amylose contents with respect to increased rates and strengths of reassociation, but use of such high temperatures is energetically unfavourable and costly. Accordingly, there is an unmet need for starches of high amylose content which can be processed below 100xc2x0 C. and still show enhanced levels of reassociation, as indicated for example by viscoamylograph measurements.
The properties of potato starch are useful in a variety of both food and non-food (paper, textiles, adhesives etc.) applications. However, for many applications, properties are not optimum and various chemical and physical modifications well known in the art are undertaken in order to improve useful properties. Two types of property manipulation which would be of use are: the controlled alteration of gelatinization and pasting temperatures; and starches which suffer less granular fragmentation during pasting than conventional starches.
Currently the only ways of manipulating the gelatinization and pasting temperatures of potato starch are by the inclusion of additives such as sugars, polyhydroxy compounds of salts (Evans and Haisman, Starke 34, 224-231, 1982) or by extensive physical or chemical pre-treatments (e.g. Stute, Starke 44, 205-214, 1992). The reduction of granule fragmentation during pasting can be achieved either by extensive physical pretreatments (Stute, Starke 44, 205-214, 1992) or by chemical cross-linking. Such processes are inconvenient and inefficient. It is therefore desirable to obtain plants which produce starch which intrinsically possesses such advantageous properties.
Starch consists of two main polysaccharides, amylose and amylopectin. Amylose is a generally linear polymer containing xcex1-1,4 linked glucose units, while amylopectin is a highly branched polymer consisting of a xcex1-1,4 linked glucan backbone with xcex1-1,6 linked glucan branches. In most plant storage reserves amylopectin constitutes about 75% of the starch content. Amylopectin is synthesized by the concerted action of soluble starch synthase and starch branching enzyme [xcex1-1,4 glucan: xcex1-1,4 glucan 6-glycosyltransferase, EC 2.4.1.18]. Starch branching enzyme (SBE) hydrolyses xcex1-1,4 linkages and rejoins the cleaved glucan, via an xcex1-1,6 linkage, to an acceptor chain to produce a branched structure. The physical properties of starch are strongly affected by the relative abundance of amylose and amylopectin, and SBE is therefore a crucial enzyme in determining both the quantity and quality of starches produced in plant systems.
In most plants studied to date e.g. maize (Boyer and Preiss, 1978 Biochem. Biophys. Res. Comm. 80, 169-175), rice (Smyth, 1988 Plant Sci. 57, 1-8) and pea (Smith, Planta 175, 270-279), two forms of SBE have been identified, each encoded by a separate gene. A recent review by Burton et al., (1995 The Plant Journal 7, 3-15) has demonstrated that the two forms of SBE constitute distinct classes of the enzyme such that, in general, enzymes of the same class from different plants may exhibit greater similarity than enzymes of different classes from the same plant. In their review, Burton et al. termed the two respective enzyme families class xe2x80x9cAxe2x80x9d and class xe2x80x9cBxe2x80x9d, and the reader is referred thereto (and to the references cited therein) for a detailed discussion of the distinctions between the two classes. One general distinction of note would appear to be the presence, in class A SBE molecules, of a flexible N-terminal domain, which is not found in class B molecules. The distinctions noted by Burton et al. are relied on herein to define class A and class B SBE molecules, which terms are to be interpreted accordingly.
However in potato, only one isoform of the SBE molecule (belonging to class B) has thus far been reported and only one gene cloned (Blennow and Johansson, 1991 Phytochem. 30, 437-444, and Koxcex2mann el al., 1991 Mol. Gen. Genet. 230, 39-44). Further, published attempts to modify the properties of starch in potato plants (by preventing expression of the single known SBE) have generally not succeeded (e.g. Mxc3xcller-Rober and Koxcex2mann 1994 Plant Cell and Environment 17, 601-613).
In a first aspect the invention provides a nucleotide sequence encoding an effective portion of a class A starch branching enzyme (SBE) obtainable from potato plants.
Preferably the nucleotide sequence encodes a polypeptide comprising an effective portion of the amino acid sequence shown in FIG. 5 (excluding the sequence MNKRIDL, which does not represent part of the SBE molecule), or a functional equivalent thereof (which term is discussed below). The amino acid sequence shown in FIG. 5 (Seq ID No. 15) includes a leader sequence which directs the polypeptide, when synthesised in potato cells, to the amyloplast. Those skilled in the art will recognise that the leader sequence is removed to produce a mature enzyme and that the leader sequence is therefore not essential for enzyme activity. Accordingly, an xe2x80x9ceffective portionxe2x80x9d of the polypeptide is one which possesses sufficient SBE activity to complement the branching enzyme mutation in E. coli KV 832 cells (described below) and which is active when expressed in E. coli in the phosphorylation stimulation assay. An example of an incomplete polypeptide which nevertheless constitutes an xe2x80x9ceffective portionxe2x80x9d is the mature enzyme lacking the leader sequence. By analogy with the pea class A SBE sequence, the potato class A sequence shown in FIG. 5 probably possesses a leader sequence of about 48 amino acid residues, such that the N terminal amino acid sequence is thought to commence around the glutamic acid residue (E) at position 49 (EKSSYN . . . etc.). Those skilled in the art will appreciate that an effective portion of the enzyme may well omit other parts of the sequence shown in the figure without substantial detrimental effect. For example, the C-terminal glutamic acid-rich region could be reduced in length, or possibly deleted entirely, without abolishing class A SBE activity. A comparison with other known SBE sequences, especially other class A SBE sequences (see for example, Burton et al, 1995 cited above), should indicate those portions which are highly conserved (and thus likely to be essential for activity) and those portions which are less well conserved (and thus are more likely to tolerate sequence changes without substantial loss of enzyme activity).
Conveniently the nucleotide sequence will comprise substantially nucleotides 289 to 2790 of the DNA sequence (Seq ID No. 14) shown in FIG. 5 (which nucleotides encode the mature enzyme) or a functional equivalent thereof, and may also include further nucleotides at the 5xe2x80x2 or 3xe2x80x2 end. For example, for ease of expression, the sequence will desirably also comprise an in-frame ATG start codon, and may also encode a leader sequence. Thus, in one embodiment, the sequence further comprises nucleotides 145 to 288 of the sequence shown in FIG. 5. Other embodiments are nucleotides 228 to 2855 of the sequence labelled xe2x80x9cpsbe2con.seqxe2x80x9d in FIG. 8, and nucleotides 57 to 2564 of the sequence shown in FIG. 12 (preferably comprising an in-frame ATG start codon, such as the sequence of nucleotides 24 to 56 in the same Figure), or functional equivalents of the aforesaid sequences.
The term xe2x80x9cfunctional equivalentxe2x80x9d as applied herein to nucleotide sequences is intended to encompass those sequences which differ in their nucleotide composition to that shown in FIG. 5 but which, by virtue of the degeneracy of the genetic code, encode polypeptides having identical or substantially identical amino acid sequences. It is intended that the term should also apply to sequences which are sufficiently homologous to the sequence of the invention that they can hybridise to the complement thereof under stringent hybridisation conditionsxe2x80x94such equivalents will preferably possess at least 85%, more preferably at least 90%, and most preferably at least 95% sequence homology with the sequence of the invention as exemplified by nucleotides 289 to 2790 of the DNA sequence shown in FIG. 5. It will be apparent to those skilled in the art that the nucleotide sequence of the invention may also find useful application when present as an xe2x80x9cantisensexe2x80x9d sequence. Accordingly, functionally equivalent sequences will also include those sequences which can hybridise, under stringent hybridisation conditions, to the sequence of the invention (rather than the complement thereof). Such xe2x80x9cantisensexe2x80x9d equivalents will preferably possess at least 85%, more preferably at least 90%, and most preferably 95% sequence homology with the complement of the sequence of the invention as exemplified by nucleotides 289 to 2790 of the DNA sequence shown in FIG. 5. Particular functional equivalents are shown, for example, in FIGS. 8 and 10 (if one disregards the various frameshift mutations noted therein).
The invention also provides vectors, particularly expression vectors, comprising the nucleotide sequence of the invention. The vector will typically comprise a promoter and one or more regulatory signals of the type well known to those skilled in the art. The invention also includes provision of cells transformed (which term encompasses transduction and transfection) with a vector comprising the nucleotide sequence of the invention.
The invention further provides a class A SBE polypeptide, obtainable from potato plants. In particular the invention provides the polypeptide in substantially pure form, especially in a form free from other plant-derived (especially potato plant-derived) components, which can be readily accomplished by expression of the relevant nucleotide sequence in a suitable non-plant host (such as any one of the yeast strains routinely used for expression purposes, e.g. Pichia spp. or Saccharomyces spp). Typically the enzyme will substantially comprise the sequence of amino acid residues 49 to 882 shown in FIG. 5 (disregarding the sequence MNKRIDL, which is not part of the enzyme), or a functional equivalent thereof. The polypeptide of the invention may be used in a method of modifying starch in vitro, comprising treating starch under suitable conditions (e.g. appropriate temperature, pH, etc.) with an effective amount of the polypeptide according to the invention.
The term xe2x80x9cfunctional equivalentxe2x80x9d, as applied herein to amino acid sequences, is intended to encompass amino acid sequences substantially similar to that shown in FIG. 5, such that the polypeptide possesses sufficient activity to complement the branching enzyme mutation in E. coli KV 832 cells (described below) and which is active in E. coli in the phosphorylation stimulation assay. Typically such functionally equivalent amino acid sequences will preferably possess at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence of the mature enzyme (i.e. minus leader sequence) shown in FIG. 5. Those skilled in the art will appreciate that conservative substitutions may be made generally throughout the molecule without substantially affecting the activity of the enzyme. Moreover, some non-conservative substitutions may be tolerated, especially in the less highly conserved regions of the molecule. Such substitutions may be made, for example, to modify slightly the activity of the enzyme. The polypeptide may, if desired, include a leader sequence, such as that exemplified by residues 1 to 48 of the amino acid sequence shown in FIG. 5, although other leader sequences and signal peptides and the like are known and may be included.
A portion of the nucleotide sequence of the invention has been introduced into a plant and found to affect the characteristics of the plant. In particular, introduction of the sequence of the invention, operably linked in the antisense orientation to a suitable promoter, was found to reduce the amount of branched starch molecules in the plant. Additionally, it has recently been demonstrated in other experimental systems that xe2x80x9csense suppressionxe2x80x9d can also occur (i.e. expression of an introduced sequence operably linked in the sense orientation can interfere, by some unknown mechanism, with the expression of the native gene), as described by Matzke and Matzke (1995 Plant Physiol. 107, 679-685). Any one of the methods mentioned by Matzke and Matzke could, in theory, be used to affect the expression in a host of a homologous SBE gene.
It is believed that antisense methods are mainly operable by the production of antisense mRNA which hybridises to the sense mRNA, preventing its translation into functional polypeptide, possibly by causing the hybrid RNA to be degraded (e.g. Sheehy et al., 1988 PNAS 85, 8805-8809; Van der Krol et al., Mol. Gen. Genet. 220, 204-212). Sense suppression also requires homology between the introduced sequence and the target gene, but the exact mechanism is unclear. It is apparent however that, in relation to both antisense and sense suppression, neither a full length nucleotide sequence, nor a xe2x80x9cnativexe2x80x9d sequence is essential. Preferably the xe2x80x9ceffective portionxe2x80x9d used in the method will comprise at least one third of the full length sequence, but by simple trial and error other fragments (smaller or larger) may be found which are functional in altering the characteristics of the plant.
Thus, in a further aspect the invention provides a method of altering the characteristics of a plant, comprising introducing into the plant an effective portion of the sequence of the invention operably linked to a suitable promoter active in the plant. Conveniently the sequence will be linked in the anti-sense orientation to the promoter. Preferably the plant is a potato plant. Conveniently, the characteristic altered relates to the starch content and/or starch composition of the plant (i.e. amount and/or type of starch present in the plant). Preferably the method of altering the characteristics of the plant will also comprise the introduction of one or more further sequences, in addition to an effective portion of the sequence of the invention. The introduced sequence of the invention and the one or more further sequences (which may be sense or antisense sequences) may be operably linked to a single promoter (which would ensure both sequences were transcribed at essentially the same time), or may be operably linked to separate promoters (which may be necessary for optimal expression). Where separate promoters are employed they may be identical to each other or different. Suitable promoters are well known to those skilled in the art and include both constitutive and inducible types. Examples include the CaMV 35S promoter (e.g. single or tandem repeat) and the patatin promoter. Advantageously the promoter will be tissue-specific. Desirably the promoter will cause expression of the operably linked sequence at substantial levels only in the tissue of the plant where starch synthesis and/or starch storage mainly occurs. Thus, for example, where the sequence is introduced into a potato plant, the operably linked promoter may be tuber-specific, such as the patatin promoter.
Desirably, for example, the method will also comprise the introduction of an effective portion of a sequence encoding a class B SBE, operably linked in the antisense orientation to a suitable promoter active in the plant. Desirably the further sequence will comprise an effective portion of the sequence encoding the potato class B SBE molecule. Conveniently the further sequence will comprise an effective portion of the sequence described by Blennow and Johansson (1991 Phytochem. 30, 437-444) or that disclosed in WO92/11375. More preferably, the further sequence will comprise at least an effective portion of the sequence disclosed in International Patent Application No. WO 95/26407. Use of antisense sequences against both class A and class B SBE in combination has now been found by the present inventors to result in the production of starch having very greatly altered properties (see below). Those skilled in the art will appreciate the possibility that, if the plant already comprises a sense or antisense sequence which efficiently inhibits the class B SBE activity, introduction of a sense or antisense sequence to inhibit class A SBE activity (thereby producing a plant with inhibition of both class A and class B activity) might alter greatly the properties of the starch in the plant, without the need for introduction of one or more further sequences. Thus the sequence of the invention is conveniently introduced into plants already having low levels of class A and/or class B SBE activity, such that the inhibition resulting from the introduction of the sequence of the invention is likely to have a more pronounced effect.
The sequence of the invention, and the one or more further sequences if desired, can be introduced into the plant by any one of a number of well-known techniques (e.g. Agrobacterium-mediated transformation, or by xe2x80x9cbiolisticxe2x80x9d methods). The sequences are likely to be most effective in inhibiting SBE activity in potato plants, but theoretically could be introduced into any plant. Desirable examples include pea, tomato, maize, wheat, rice, barley, sweet potato and cassava plants. Preferably the plant will comprise a natural gene encoding an SBE molecule which exhibits reasonable homology with the introduced nucleic acid sequence of the invention.
In another aspect, the invention provides a plant cell, or a plant or the progeny thereof, which has been altered by the method defined above. The progeny of the altered plant may be obtained, for example, by vegetative propagation, or by crossing the altered plant and reserving the seed so obtained. The invention also provides parts of the altered plant, such as storage organs. Conveniently, for example, the invention provides tubers comprising altered starch, said tubers being obtained from an altered plant or the progeny thereof. Potato tubers obtained from altered plants (or the progeny thereof) will be particularly useful materials in certain industrial applications and for the preparation and/or processing of foodstuffs and may be used, for example, to prepare low-fat waffles and chips (amylose generally being used as a coating to prevent fat uptake), and to prepare mashed potato (especially xe2x80x9cinstantxe2x80x9d mashed potato) having particular characteristics.
In particular relation to potato plants, the invention provides a potato plant or part thereof which, in its wild type possesses an effective SBE A gene, but which plant has been altered such that there is no effective expression of an SBE A polypeptide within the cells of at least part of the plant. The plant may have been altered by the method defined above, or may have been selected by conventional breeding to be deleted for the class A SBE gene, presence or absence of which can be readily determined by screening samples of the plants with a nucleic acid probe or antibody specific for the potato class A gene or gene product respectively.
The invention also provides starch extracted from a plant altered by the method defined above, or the progeny of such a plant, the starch having altered properties compared to starch extracted from equivalent. but unaltered, plants. The invention further provides a method of making altered starch, comprising altering a plant by the method defined above and extracting therefrom starch having altered properties compared to starch extracted from equivalent, but unaltered, plants. Use of nucleotide sequences in accordance with the invention has allowed the present inventors to produce potato starches having a wide variety of novel properties.
In particular the invention provides the following: a plant (especially a potato plant) altered by the method defined above, containing starch which, when extracted from the plant, has an elevated endotherm peak temperature as judged by DSC, compared to starch extracted from a similar, but unaltered, plant; a plant (especially a potato plant) altered by the method defined above, containing starch which, when extracted from the plant, has an elevated viscosity onset temperature (conveniently elevated by 10-25xc2x0 C.) as judged by viscoamylograph compared to starch extracted from a similar, but unaltered, plant; a plant (especially a potato plant) altered by the method defined above, containing starch which, when extracted from the plant. has a decreased peak viscosity (conveniently decreased by 240-700 SNUs) as judged by viscoamylograph compared to starch extracted from a similar, but unaltered, plant; a plant (especially a potato plant) altered by the method defined above, containing starch which, when extracted from the plant, has an increased pasting viscosity (conveniently increased by 37-260 SNUs) as judged by viscoamylograph compared to starch extracted from a similar, but unaltered, plant; a plant (especially a potato plant) altered by the method defined above, containing starch which, when extracted from the plant, has an increased set-back viscosity (conveniently increased by 224-313 SNUs) as judged by viscoamylograph compared to starch extracted from a similar, but unaltered, plant; a plant (especially a potato plant) altered by the method defined above, containing starch which, when extracted from the plant, has a decreased set-back viscosity as judged by viscoamylograph compared to starch extracted from a similar, but unaltered, plant; and a plant (especially a potato plant) altered by the method defined above, containing starch which, when extracted from the plant, has an elevated amylose content as judged by iodometric assay (i.e. by the method of Morrison and Laignelet 1983, cited above) compared to starch extracted from a similar, but unaltered, plant. The invention also provides for starch obtainable or obtained from such plants as aforesaid.
In particular the invention provides for starch which, as extracted from a potato plant by wet milling at ambient temperature, has one or more of the following properties, as judged by viscoamylograph analysis performed according to the conditions defined below: viscosity onset temperature in the range 70-95xc2x0 C. (preferably 75-95xc2x0 C.); peak viscosity in the range 500-12 stirring number units; pasting viscosity in the range 214-434 stirring number units; set-back viscosity in the range 450-618 or 14-192 stirring number units; or displays no significant increase in viscosity during viscoamylograph. Peak, pasting and set-back viscosities are defined below. Viscosity onset temperature is the temperature at which there is a sudden, marked increase in viscosity from baseline levels during viscoamylograph, and is a term well-known to those skilled in the art.
In other particular embodiments, the invention provides starch which as extracted from a potato plant by wet milling at ambient temperature has a peak viscosity in the range 200-500 SNUs and a set-back viscosity in the range 275-618 SNUs as judged by viscoamylograph according to the protocol defined below; and starch which as extracted from a potato plant by wet milling at ambient temperature has a viscosity which does not decrease between the start of the heating phase (step 2) and the start of the final holding phase (step 5) and has a set-back viscosity of 303 SNUs or less as judged by viscoamylograph according to the protocol defined below.
For the purposes of the present invention, viscoamylograph conditions are understood to pertain to analysis of a 10% (w/w) aqueous suspension of starch at atmospheric pressure, using a Newport Scientific Rapid Visco Analyser with a heating profile of: holding at 50xc2x0 C. for 2 minutes (step 1), heating from 50 to 95xc2x0 C. at a rate of 1.5xc2x0 C. per minute (step 2), holding at 95xc2x0 C. for 15 minutes (step 3), cooling from 95 to 50xc2x0 C. at a rate of 1.5xc2x0 C. per minute (step 4), and then holding at 50xc2x0 C. for 15 minutes (step 5). Peak viscosity may be defined for present purposes as the maximum viscosity attained during the heating phase (step 2) or the holding phase (step 3) of the viscoamylograph. Pasting viscosity may be defined as the viscosity attained by the starch suspensions at the end of the holding phase (step 3) of the viscoamylograph. Set-back viscosity may be defined as the viscosity of the starch suspension at the end of step 5 of the viscoamylograph.
In yet another aspect the invention provides starch from a potato plant having an apparent amylose content (% w/w) of at least 35%, as judged by iodometric assay according to the method described by Morrison and Laignelet (1983 J. Cereal Science 1, 9-20). The iodometric assay is conducted by dissolving the starch in urea-dimethylsuphoxide (xe2x80x9cUDMSOxe2x80x9d), and aliquots of the solution are used to determine total amylose (measured on lipid-free starch, precipitated from urea-dimethylsulphoxide solution with ethanol). UDMSO may be obtained by mixing 9 volumes of dimethylsulphoxide with 1 volume of 6-M urea. Aliquots of the starch-UDMSO solution are then treated with 12xe2x80x94KI reagent (2 mg I2, 20 mg KI/ml) at different concentrations at constant temperature and followed via colorimetry in order to determine the Blue Value. The Blue Value is defined as the absorbance/cm at 635 nm of 10 mg anhydrous starch in 100 ml dilute I2xe2x80x94KI solution at 20xc2x0 C. Amylose content is calculated from the Blue Value according to the regression equation: amylose (%)=(28.414xc3x97Blue Value)xe2x88x926.218. Preferably the starch will have an amylose content of at least 40%, more preferably at least 50%, and most preferably at least 66%. Starch obtained directly from a potato plant and having such properties has not hitherto been produced. Indeed, as a result of the present invention, it is now possible to generate in vivo potato starch which has some properties analogous to the very high amylose starches (e.g. HYLON(copyright) VII starch) obtainable from maize.
Starches with high (at least 35%) amylose contents find commercial application as, amongst other reasons, the amylose component of starch reassociates more strongly and rapidly than the amylopectin component during retrogradation processes. This may result, for example, in pastes with higher viscosities, gels of greater cohesion, or films of greater strength for starches with high (at least 35%) compared with normal (less than 35%) amylose contents. Alternatively, starches may be obtained with very high amylose contents, such that the granule structure is substantially preserved during heating, resulting in starch suspensions which demonstrate substantially no increase in viscosity during cooking (i.e. there is no significant viscosity increase during viscoamylograph conditions defined above). Such starches typically exhibit a viscosity increase of less than 10% (preferably less than 5%) during viscoamylograph under the conditions defined above.
In commerce, these valuable properties are currently obtained from starches of high amylose content derived from maize plants. It would be of commercial value to have an alternative source of high amylose starches from potato as other characteristics such as granule size, organoleptic properties and textural qualities may distinguish application performances of high amylose starches from maize and potato plants.
Thus high amylose starch obtained by the method of the present invention may find application in many different technological fields, which may be broadly categorised into two groups: food products and processing; and xe2x80x9cIndustrialxe2x80x9d applications. Under the heading of food products, the novel starches of the present invention may find application as, for example, films, barriers, coatings or gelling agents. In general, high amylose content starches absorb less fat during frying than starches with low amylose content, thus the high amylose content starches of the invention may be advantageously used in preparing low fat fried products (e.g. potato chips, crisps and the like). The novel starches may also be employed with advantage in preparing confectionery and in granular and retrograded xe2x80x9cresistantxe2x80x9d starches. xe2x80x9cResistantxe2x80x9d starch is starch which is resistant to digestion by xcex1-amylase. As such, resistant starch is not digested by xcex1-amylases present in the human small intestine, but passes into the colon where it exhibits properties similar to soluble and insoluble dietary fibre. Resistant starch is thus of great benefit in foodstuffs due to its low calorific value and its high dietary fibre content. Resistant starch is formed by the retrogradation (akin to recrystallization) of amylose from starch gels. Such retrogradation is inhibited by amylopectin. Accordingly, the high amylose starches of the present invention are excellent starting materials for the preparation of resistant starch. Suitable methods for the preparation of resistant starch are well-known to those skilled in the art and include, for example, those described in U.S. Pat. Nos. 5,051,271 and 5,281,276. Conveniently the resistant starches provided by the present invention comprise at least 5% total dietary fibre, as judged by the method of Prosky et al., (1985 J. Assoc. Off. Anal. Chem. 68, 677), mentioned in U.S. Pat. No. 5,281, 276.
Under the heading of xe2x80x9cIndustrialxe2x80x9d applications, the novel starches of the invention may be advantageously employed, for example, in corrugating adhesives, in biodegradable products such as loose fill packaging and foamed shapes, and in the production of glass fibers and textiles.
Those skilled in the art will appreciate that the novel starches of the invention may, if desired, be subjected in vitro to conventional enzymatic, physical and/or chemical modification, such as cross-linking, introduction of hydrophobic groups (e.g. octenyl succinic acid, dodecyl succinic acid), or derivatization (e.g. by means of esterification or etherification).
In yet another aspect the invention provides high (35% or more) amylose starches which generate paste viscosities greater than those obtained from high amylose starches from maize plants after processing at temperatures below 100xc2x0 C. This provides the advantage of more economical starch gelatinization and pasting treatments through the use of lower processing temperatures than are currently required for high amylose starches from maize plants.